I've had luck using 8 cutters (eg NotI, SfiI, PacI, PmeI, AscI, etc.)
lambda terminase (we got it from Epicenter) and a FIGE (Field Inversion
Gel Electrophoresis) inverter (a PPI-200 from CBS Scientific). Then
it's just like doing a normal plasmid restriction map. It is critical
to recirculate the buffer (I just use 0.5xTBE with EtBr at 200 ug/liter
in both the gel and the running buffer) and not to overload the gel. A
CHEF gel seems to sieve big bright bands fine, but (in my hands) a FIGE
gel will not. I like FIGE over CHEF, because I can hook a FIGE
inverter to any gel box with recirculation ports. That means I can run
40+ slot gels. Also, it's no big deal to take a picture of a gel and
then continue running it with FIGE--I don't think that will work with
most CHEF gel rigs. Size standards are a little tough, also. I use the
"8-48" kb standard and the 5 kb ladder (from BioRad). Lambda ladders
would be nice, but they always come embedded in agarose and this did
not seem compatible with FIGE. (Note, I don't embed any of my
reactions--I just avoid shearing them as much as possible.)
Roxanne Walder wrote:
> What is the best method for restriction mapping BAC and/or PAC DNA?